Research in Plant Biology 2019, 9: 9-15 doi: 10.25081/ripb.2019.v9.3779 http://updatepublishing.com/journal/index.php/ripb Research Article Micropropagation, genetic fidelity assessment and phytochemical studies of Clerodendrum thomsoniae ISSN: 2231-5101X Balf. f. with special reference to its anti-stress properties Pallab Kar1, Arnab Kumar Chakraborty1, Malay Bhattacharya2, Tanmayee Mishra2 and Arnab Sen1* 1Molecular Genetics Laboratory, Department of Botany, University of North Bengal, Siliguri- 734013, India, 2Molecular Biology and Tissue Culture Laboratory, Department of Tea Science, University of North Bengal, Siliguri- 734013, India ABSTRACT Clerodendrum thomsoniae commonly known as bleeding heart vine or bag flower. In this study, in-vitro callus regeneration of C. thomsoniae through nodal culture has been tried. Murashige and Skoog’s medium (MS) with BAP was found suitable for shoot and root development. To detect clonal fidelity in C. thomsoniae, RAPD and ISSR markers were used. Ten RAPD decamers produced 65 amplicons, while ten ISSR primers generated 75 bands in both in-vitro plantlets and Received: February 01, 2019 mother plants. The amplified products of parent plants and the regenerated plants were found to be monomorphic in Accepted: March 03, 2019 RAPD and ISSR analyses. A number of compounds with potential therapeutic and biological activity had been detected Published: March 08, 2019 with the help of GC-MS analysis. Among this the compounds namely 3-Hydroxybutyric acid, Azelaic acid, Linoleic acid, Oleic acid, Squalene, Stigmasterol are the important compounds mainly responsible for anti-cancer, anti-inflammatory, antiasthma, antimicrobial, antistress activity and so on. Taking into account of all the GC-MS and in-silico molecular *Corresponding Author: docking data, it can be concluded that some of these compounds may be potential to future drug industry. Arnab Sen Email: senarnab_nbu@ hotmail.com KEYWORDS: Clerodendrum thomsoniae, RAPD, ISSR, nodal stem segment, genetic fidelity, GC-MS INTRODUCTION Clerodendrum thomsoniae is a well known cultivated ornamental plant with beautiful flowers and has been extensively used in the India has a long tradition of growing ornamental flowers that horticulture as pot plants for its attractive flowers. The species is have recently been exploited both in domestic and international relatively new to the floriculture industry because unavailability market [1]. However, due to the never ending search of the and lesser number of seed production. C. thomsoniae is a customers for new flowering plants lead the floriculture rambling, twining, vine like shrub native to tropical West industries to explore the new species. Bleeding heart vine Africa. Bleeding-heart vine or Bag-flower is the common name (Clerodendrum thomsoniae) belonging to the family Lamiaceae, of Clerodendrum thomsoniae [5,4]. is a potent ornamental plant recently exploited by the floriculture Apart from having immense floriculture and horticulture industries [2]. Floriculture industry mainly depends on two importance, this plant has been utilized in traditional medicine. important components, firstly, trade of flowering inflorescences The other members of the genus Clerodendrum like C. indicum, and type of foliage, and secondly, supply of nursery stocks C. viscosum, C. serratum, C. colebrookianum etc. are medicinal such as tubers, seeds, bulbs, cutting-raised plants and tissue and used as folk medicine [6-9]. The leaves and flowers of culture raised plantlets [3].Propagation through conventional C. thomsoniae are the main sources of several medicinally techniques have some limitations and cannot fulfill the high important phytocompounds. These phytocompounds protect demand of ornamental plants, therefore, there is in need of human body from oxidative stress by its own competent defense urgent attention for in-vitro multiplication [4]. mechanism and curing diversified diseases like bruises, cuts, skin Copyright: © 2019 The authors. This article is open access and licensed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) which permits unrestricted, use, distribution and reproduction in any medium, or format for any purpose, even commercially provided the work is properly cited. Attribution — You must give appropriate credit, provide a link to the license, and indicate if changes were made. Res Plant Biol • 2019 • Vol 9 9 Kar, et al. rashes and sores etc. [10]. Oxidative stress induced by reactive Table 1: Effect of sucrose on regeneration of shoot buds from oxygen and nitrogen species (ROS and RNS) is a major causative callus of C. thomsoniae agent in the induction of several brain disorders including BAP (mg/l) NAA (mg/l) Sucrose (%) Mean no. of shoots/callus neurogenerative diseases [11] and depression [12]. 1 mg/l 0.5 mg/l 1 2.76±0.39 2 3.71±0.25 Apart from some studies on propagation, plant growth 3 6.5±0.5 retardants etc., no attempt so far has been made to regenerate 2 mg/l 0.5 mg/l 1 6.98±0.21 2 9.07±0.17 C. thomsoniae through in-vitro methods. However, proper 3 13±1.5 molecular characterization and profiling of phytochemical 3 mg/l 0.5 mg/l 1 7.54±0.24 constituents of C. thomsoniae still remains largely obscure. So, 2 8.24±0.30 initiatives were taken to explore molecular biology and medicinal 3 9.5±0.5 property detection through GC-MS. Keeping all these lacunae in 4 mg/l 0.5 mg/l 1 1.31±0.21 2 2.5±0.10 mind, the aim of the present study is to standardize the working 3 4±1.0 protocol of regeneration of C. thomsoniae by way of regeneration through callus culture, detection of somaclonal variations, if any and medicinal characterization of both the mother plant Hardening and Transfer of Plants to Soil and tissue culture raised plantlets. Within the same scope we will try to evaluate whether the bioactive compounds present After 4 weeks of in-vitro root development, the plantlets were carefully in C. thomsoniae could be responsible for reducing depression taken out of the culture bottle without causing much damage to the with an in-silico molecular docking approach. plant parts. The roots were washed gently under running tap water to completely remove the medium. Then the plantlets were transferred MATERIALS AND METHODS to a mixture of sandy soil and farm yard manure with a ratio of 1:1 (v/v). Hardening of plantlets was conducted for 30 days in green Establishment of Aseptic Culture house condition and was finally transferred to the field. The survival percentage of the acclimatized plantlets was recorded. The nodal segments of C. thomsoniae were collected from our laboratory garden for aseptic culture. The explants were washed Molecular Analysis in 1% tween 20 for 30 minutes and then rinsed several times with double distilled water (DDW). The explants were then The genomic DNA of field grown plant and the in-vitro propagated plantlets was extracted using Genelute Plant surface sterilized with 0.1% HgCl for 5 min and then rinsed 2 Genomic DNA kit (Sigma, St. Louis, MI, USA, Cat# G2N-70). several times with sterile DDW. Explants were finally treated Based on the spectrophotometric analysis, the DNA was diluted with 70% alcohol for 45 seconds and washed several times with to a concentration of 25 ng/μl. Initially, a total of 20 RAPD and sterile DDW to remove the traces of alcohol. The explants were 15 ISSR primers were screened for PCR amplification. Both then trimmed to approximately 3 cm. in length and blot dried. RAPD and ISSR amplifications were performed using 25 μL of All these operations were conducted under aseptic environment PCR mixture containing 12.5 μl PCR Master Mix 2X (Promega, of laminar air flow cabinet. Cat# M7122), 1.25 μl of primer (0.25 μm), 2 μl of template DNA (25 ng/μl) and Pyrogen-free water to a final volume of 25 μl. Callus Culture and Plantlet Regeneration The PCR reactions were performed on an Applied Biosystems Thermocycler 2720. The amplification condition of RAPD was The trimmed explants were aseptically inoculated in MS 94 °C for 4 min, followed by 40 cycles of amplification with 1 min medium [13] and Woody Plant medium [14], containing denaturation at 94 °C, 1 min annealing at 37 °C, 2 min primer various concentration of growth regulators (BAP and NAA) extension at 72 °C, and a final extension at 72 °C for 10 min. along with three different concentrations of sucrose (Table 1) The conditions of the thermal cycle for ISSR amplifications were and 0.8% agar. The pH of the media was adjusted to 5.6-5.8 94 °C for 5 min, followed by 35 cycles of amplification having before adding agar and autoclaved at 121ºC for 20 min at 15 psi. 45 s for denaturation at 94 °C, 1 min annealing at 52 °C, 1 min primer extension at 72 °C, and a final extension at 72 °C for Different concentrations of cytokinins like BAP (6-benzyl amino 7 min. The PCR products were electrophoresed on 1.5% (w/v) purine) and auxins like NAA (1-napthaleneacetic acid) were agarose gel with two DNA markers- λ DNA/EcoRI/HindIII double used in this study for initiation of callus and shoot. Controlled digest (Chromous Biotech, Cat# MAN 06) and a 100 base pair cultures with no hormone were also prepared. Sub-culturing was (bp) DNA ladder (Chromous Biotech, Cat# LAN 02), and were done at 2 weeks interval in the same media having the same photographed with Gel Documentation system (UVi). hormonal composition [15]. BAP was applied at the rate of 1, 2, 3 and 4 mg/l and NAA at the rate of 0.5mg/l to observe various GC-MS Analysis stages of in-vitro callusing and subsequent shoot formation. The cultures were incubated under 2000-3000 lux light intensity for The bio-active compounds of the mother and tissue cultured 16 h/day at 25° ± 2° C temperature.